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Background/aim: We evaluated the relationship between low bone mineral density (BMD), also called osteopenia, and prognosis in patients who underwent resection for pancreatic cancer (PC)., Patients and Methods: We enrolled 91 consecutive patients who underwent curative resections for PC between May 2009 and January 2019. Their BMDs were measured at the Th11 vertebra using computed tomography. Patients were then divided by age-adjusted standard BMD values into the osteopenia group (n=34) and the non-osteopenia group (n=57). Their overall survival (OS) and recurrence-free survival (RFS) were compared (log-rank test)., Results: The two groups did not differ in age, BMI, tumor marker, operation time, blood loss, postoperative complications or stage. The osteopenia group had significantly worse 3-year rates for OS (46% vs. 30%, p=0.04) and RFS (41% vs. 26%, p=0.01). In multivariate analysis, osteopenia was an independent prognostic factor for RFS (HR=2.16, p=0.01)., Conclusion: Osteopenia is an adverse prognostic factor for patients with resected PC., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: The safety and efficacy of laparoscopic total gastrectomy (LTG) for remnant gastric cancer (RGC) remains unclear. The purpose of this study was to compare the clinical outcomes of LTG with open total gastrectomy (OTG) for RGC., Patients and Methods: Twenty-two patients who underwent total gastrectomy for RGC were enrolled in this study., Results: LTG was carried out in seven patients, and OTG was performed in the remaining 15 patients. The mean operation time in the LTG group was longer than that in the OTG group. The estimated blood loss in the LTG group was less than that in the OTG group. No cases in the LTG group required open conversion. Postoperatively, the first meal and defecation were earlier in the LTG group than in the OTG group. The overall survival rates of the two groups were comparable., Conclusion: Laparoscopic total gastrectomy is a feasible surgical option for RGC., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: The mouse vagina exhibits stratified squamous epithelium, which is comprised of multiple cell layers. We previously showed that erbB signaling, induced by epithelial estrogen receptor 1 (ESR1), is required for the initial differentiation of the epithelium. However, the downstream effector that mediates terminal differentiation in the apical layers remains elusive. The contribution of fibroblast growth factor (FGF) to vaginal epithelial cell differentiation was investigated., Materials and Methods: Vaginas from wild-type or epithelium-specific Esr1 conditional knockout (cKO) mice were analyzed using immunohistochemistry and quantitative real-time RT-PCR., Results: Of the FGF ligands examined, Fgf22 mRNA was significantly induced following estrogen treatment. Furthermore, FGF downstream signaling, phosphorylated FRS2 and ERK1/2 were exclusively expressed in the apical layers of the vaginal epithelium. No changes in such expression were observed in the Esr1 cKO mice., Conclusion: FGF-ERK/MAPK pathway may be a main inducer of terminal differentiation in the mouse vaginal epithelium., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Perinatal diethylstilbestrol (DES) treatment induces the polyovular follicle containing two or more oocytes in a follicle of mouse ovary through estrogen receptor (ER) β. The aim of the study was to investigate the direct effects of DES on the neonatal mouse ovary and the gene expression of activins., Materials and Methods: Ovaries from neonatal wild-type (WT) or ERβ- knockout (ERβKO) mice were organ-cultured in a serum-free medium with or without DES, and polyovular follicle induction and expression of activin signaling related genes were examined., Results: The polyovular follicle and cyst incidence in DES-treated organ-cultured ovaries from WT mice, but not from ERβKO mice, was significantly higher than that of control non-treated cultures. DES altered inhibin (Inh) a, Inhba and Inhbb expression in organ-cultured ovaries from C57BL/6J mice, while no change in Inha and an increase of Inhbb were observed by DES, in both WT and ERβKO mice., Conclusion: Alterations in activin signaling are involved in the polyovular follicle induction by DES., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Theca cells produce androgen by 17α-hydroxylase-17,20-lyase encoded by Cyp17a1, and conversion of androgen to estrogen in granulosa cells is regulated by gonadotropins. Women with polycystic ovarian syndrome (PCOS) exhibit elevated levels of androgens due to high Cyp17a1 expression and alterations in gene expression in granulosa cells. The aim of this study was to examine the interaction between theca and granulosa cells in PCOS-model mice., Materials and Methods: To produce PCOS-model mice, neonatal mice were injected with 1 μg TP for 3 days from the day of birth. Gonadotropins were injected according to the superovulation protocol to 3-month-old control mice and PCOS-model mice. Histological changes and expression of genes involved in steroidogenesis, ovulation and luteinization were investigated by immunohistochemistry and real-time RT-PCR, respectively., Results: Pregnant mare serum gonadotropin (PMSG) induced the expression of genes involved in steroidogenesis in control prepubertal mice, whereas human chorionic gonadotropin (hCG) reduced Cyp17a1 expression and induced phospho-ERK1/2 in granulosa cells. Cyp17a1 was reduced in PMSG-primed PCOS-model mice regardless of hCG injection, and PMSG induced phosphorylation of ERK1/2 in granulosa cells., Conclusion: Phospho-ERK1/2 in granulosa cells can be correlated with reduced Cyp17a1 expression in theca cells, and the interaction between granulosa and theca cells may be impaired in PCOS-model mice., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Neonatal diethylstilbestrol (DES) treatment induces polyovular follicles (PFs), which contain more than two oocytes in a follicle, through estrogen receptor (ER) β, not ERα. 2,3-Bis(4-hydroxyphenyl)-propionitrile (DPN) is a specific ERβ agonist; the effects of neonatal DPN exposure on PF induction and gene expression in the mouse ovary were examined., Materials and Methods: Histological analysis and real-time reverse transcription-polymerase chain reaction were performed., Results: The PF incidence was significantly high in the ovary of neonatally DPN-exposed mice compared to that in oil-exposed mice. The gene expression of growth differentiation factor 9 (Gdf9), Mullerian-inhibiting substance, steroidogenic factor 1 (Sf1) and steroidogenic acute regulatory protein (Star) in the ovary was significantly increased in the mice neonatally exposed to 40 μg DPN compared to oil-treated mice., Conclusion: Since SF1 is an important transcription factor of several genes involved in ovarian function, up-regulation of Sf1 expression by neonatal exposure to DPN, through ERβ, might affect expression of Gdf9, Mis and Star, resulting in increased PFs in mouse ovary., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: In neonate mice exposed to diethylstilbestrol (neoDES), vaginal epithelium shows persistent proliferation and stratification even after ovariectomy. Tissue recombination studies suggest that neonatally-estrogenized vaginal stroma can induce vaginal epithelial hyperplasia depending on the stromal age. This study examined the proliferative effect of the vaginal stroma from 8-day-old mice treated with DES on the vaginal epithelia of 8-day-old and adult mice., Materials and Methods: Vaginal epithelium and stroma from 8-day-old and adult mice was recombined, and grafted to ovariectomized host mice., Results: The 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in the epithelium and the number of epithelial cell layers were not significantly different between epithelia from 8-day-old and adult mice when combined with stroma from 8-day-old control mice. BrdU-labeled cells in the vaginal epithelia from both age groups combined with the stroma from 8-day-old neoDES mice exhibited higher values. The epithelium from neoDES adult mice had a lower percentage of BrdU-labeled cells., Conclusion: The stroma from 8-day-old neoDES mice induces epithelial cell proliferation, but lower stromal cell proliferation.

Amphibian eggs spawned in water are exposed immediately to various chemicals present in their water. The present study aimed to investigate the accumulation and pharmacokinetics of 17α-ethynylestradiol (EE(2)), bisphenol A (BPA), and nonylphenol (NP), as well as 17β-estradiol (E(2)), in the pre-hatch and post-hatch embryos of the frog Rana rugosa. Fertilized eggs were exposed to chemicals at a final concentration of 500 nM in breeding water for two days, then the embryos with jelly coats were reared in fresh-breeding water without supplementation of the xenoestrogens for six more days. All exogenous chemicals were concentrated in the embryo body at two days after fertilization, whereas their concentrations in the jelly coat were the same as those in the breeding water. The bioconcentration factors for E(2), EE(2), BPA, and NP were 217.9, 170.2, 382.3, and 289.1, respectively, suggesting that the estrogenic chemicals were concentrated in the embryo body through the jelly coat.

Estrogen regulates morphological changes in reproductive organs, such as the vagina and uterus, during the estrous cycles in mice. Estrogen depletion by ovariectomy in adults results in atrophy accompanied by apoptosis in vaginal and uterine cells, while estrogen treatment following ovariectomy elicits cell proliferation in both organs. Sequential changes in mRNA expression of wingless-related MMTV integration site (Wnt) and Notch signaling genes were analyzed in the vagina and uterus of ovariectomized adult mice after a single injection of 17β-estradiol to provide understanding over the molecular basis of differences in response to estrogen in these organs. We found estrogen-dependent up-regulation of Wnt4, Wnt5a and p21 and down-regulation of Wnt11, hairy/enhancer-of-split related with YRPW motif-1 (Hey1) and delta-like 4 (Dll4) in the vagina, and up-regulation of Wnt4, Wnt5a, Hey1, Heyl, Dll1, p21 and p53 and down-regulation of Wnt11, Hey2 and Dll4 in the uterus. The expression of Wnt4, Hey1, Hey2, Heyl, Dll1 and p53 showed different patterns after the estrogen injection. Expression patterns for Wnt5a, Wnt11, Dll4 and p21 in the vagina and uterus were similar, suggesting that these genes are involved in the proliferation of cells in both those organs in mice.

Background: Cigarette smoke contains carcinogenic aromatic and heterocyclic amines that are metabolized by N-acetyltransferase (NAT). These carcinogens also produce reactive oxygen species that are metabolized by manganese superoxide dismutase (MnSOD). The association between prostate cancer (PCA) and the polymorphism of MnSOD and NAT, and cigarette smoking was investigated., Patients and Methods: DNA samples from 187 PCA patients and 175 age-matched controls were genotyped for MnSOD, NAT1 and NAT2 by PCR restriction fragment length polymorphism analysis and DNA sequencing., Results: MnSOD AA genotype, as compared to MnSOD VV and VA, was associated with PCA (odds ratio, 1.65; 95% confidence interval, 1.03-2.66. There was no association of PCA with NAT or smoking. Results of exploratory analyses of the data suggest that the association of PCA and MnSOD exists only in the subpopulation of rapid NAT1 genotypes and smokers., Conclusion: The present study demonstrates the association of PCA and MnSOD. Oxidative stress and cigarette smoking may play an important role in the carcinogenesis of the prostate in those who have MnSOD AA and rapid NAT1 genotypes.

Reproductive organs in female mice are susceptible to exposure to estrogenic substances especially during development. In the present study, C57BL/6J female mice were exposed to the synthetic estrogens ethinylestradiol (EE2) or diethylstilbestrol (DES), 10-100 or 6.7-67 microg/kg bw, respectively, in utero from day 10 to 18 of pregnancy, and their effects were analyzed at 30 and 40 days of age. Both EE2 and DES reduced the survival rate of fetuses and newborns in a dose-dependent manner. Polyovular follicles (PF) were found in the ovaries of all groups at 30 days of age including oil-injected controls. However, the incidence of PF was significantly higher in the 50 microg/kg EE2- and 33.3 microg/kg DES-exposed mice than the control. In vaginal epithelia of the in utero EE2 exposed, ovariectomized mice, stratification and cornification were encountered even 10 days after ovariectomy. Especially, vaginae in the ovariectomized mice given high dose of EE2 or DES in utero showed ovary-independent proliferation of the epithelium. Thus, it is clear that prenatal exposure to EE2 or DES induces reproductive abnormalities, including PF, ovary-independent vaginal epithelial stratification and cornification.

Proliferation of the vagina and uterus in rodents is stimulated by ovarian estrogen, and ovariectomy induces regression of these tracts. In mouse, reproductive tracts, organ weights, vaginal epithelial thickness, the number of epithelial cell layers, and uterine epithelial cell height were significantly reduced 1 to 20 days after ovariectomy when compared with those of intact, mice taken at the estrous stage. No significant differences in those parameters were found between ovariectomized mice implanted with estradiol and intact estrous mice. Vaginal epithelium at estrus was composed of 10-15 layers of epithelial cells with superficial keratinized layers, however, the vaginal epithelium began to regress one day after ovariectomy. Infiltration of leukocytes was encountered in the vaginal epithelium 2-3 days after ovariectomy. Protein expression in the regressing vagina and uterus after ovariectomy was examined by two-dimensional polyacrylamide gel electrophoresis. After ovariectomy, new proteins appeared in the regressing vagina, but not in the regressing uterus. Expressions of 7 and 6 proteins in the vagina and uterus, respectively, reached peak levels 2-3 days after ovariectomy. The expression of ovariectomy-specific proteins and infiltration of leukocytes in vaginal epithelium was inhibited by the injection of cycloheximide, an inhibitor of protein synthesis. These results suggest that some proteins participate in the regression of mouse reproductive tracts after estrogen withdrawal induced by ovariectomy.

Development and mitotic activity of metrial gland in rat uterus were investigated focusing on the point of implantation during mid-pregnancy. In decidua basalis, the granulated metrial gland (GMG) cells slightly increased in number during days 8-12 of pregnancy and then decreased, while the cells appearing in the mesometrial triangle on day 9 continued to increase until day 16, forming the metrial gland in situ. By contrast, the mitotic change in the mesometrial triangle was similar to that in the other 2 regions, decidua basalis and the inner layer of myometrium. In all 3 regions, mitotic rate rose drastically on day 9, and then gradually decreased until day 16. However, most of the mitotic cells lacked the PAS-positive cytoplasmic granules in any regions. Mitotic activity in GMG cells was very low and almost unchanged throughout the gland development. These results suggest that the rapid rise of mitotic rate is due to the active mitosis of PAS-negative cells, presumably GMG precursor cells, existing widely in the implantation point of rat uterus during the early development of the gland. Immunocytochemical study revealed that progesterone receptors were localized in decidual and muscle cells and fibroblasts, but not in GMG cells during the development of the gland. The present study suggests that the metrial gland cells are proliferated, differentiated and maintained by the progesterone-sensitive stromal cells, such as decidual cells and/or fibroblasts.

Neonatal treatment of female mice with a synthetic estrogen, diethylstilbestrol (DES), results in adenosis, ovary-independent epithelial proliferation and cornification, and downgrowths in the vagina. Protein synthesis was examined in the vagina of 45-day-old, ovariectomized C57BL/Tw mice which had been given 5 daily injections of 2 micrograms DES or the oil vehicle alone from the day of birth, and in those of age-matched, ovariectomized mice given 3 daily injections of 0.1 microgram DES from 42 days of age. [35S]Methionine-labeled proteins of the vagina were analyzed by two-dimensional polyacrylamide gel electrophoresis. Major changes were observed in 11 proteins of the vagina in both neonatally and postnatally DES-exposed groups. Six of the 11 proteins were increased in expression, but one of the remaining proteins was decreased. In the group of neonatally DES-exposed mice alone, expressions of 4 proteins (MW. 128, 90, 46 and 44 kDa) were markedly increased. These results indicate that neonatal exposure of mice to DES induced ovary-independent, persistent alteration in the protein synthesis of the vagina.

Postpubertal progesterone injections slightly inhibited the proliferation and cornification of the vaginal epithelium induced by neonatal injections of diethylstilbestrol (DES). In vaginae of neonatally DES-exposed mice (DES mice), 9 protein expressions (PEX) appeared newly; 13 PEX decreased; 5 PEX increased compared to those in the controls. In vaginae of DES mice, PEX were altered by postpubertal injections of progesterone (DES-P); 3 PEX disappeared; 11 PEX were reversed to those in the controls. Progesterone injections impaired the proliferation of the vaginal epithelium, reversing 11 PEX compared to those in DES mice. In uteri of DES-P mice, 3 PEX increased, 2 PEX decreased, 2 PEX appeared and 1 PEX disappeared compared to those in DES mice. In conclusion, it was shown that postpubertal injections of progesterone alter the protein expression in the vagina and uterus of DES mice.

Postpubertal injections of a synthetic estrogen, diethylstilbestrol (-DES), and an anti-estrogen, tamoxifen (-Tx), stimulate proliferation of vaginal and uterine epithelial cells of ovariectomized (OVX) adult mice. In vaginae of two groups of OVX mice treated neonatally with DES (DES-) and oil vehicle alone (Oil-), postpubertal injections of DES altered all and 25 out of 37 protein expressions examined, except for keratin polypeptides, respectively. Twenty-one of the DES- and Tx-altered protein expressions showed the same behavior, suggesting that Tx acts as an estrogen agonist on these proteins. Both neonatally Oil- and DES-treated OVX mice given postpubertal injections of DES (Oil-DES and DES-DES) and Tx (Oil-Tx and DES-Tx) showed similar histological changes in the vagina. Epithelial proliferation and superficial cornification were observed in vaginae of five groups of Oil-DES, Oil-Tx, DES-DES, DES-Tx and DES-Oil (given oil postpubertally) OVX mice. In these groups, 13 proteins showed the same behavior, implying that these proteins are related to proliferation and cornification of mouse vagina. The histology of the uterus in DES-Oil OVX mice closely resembled that in Oil-Oil OVX mice; in protein expressions of the uterus, 5 proteins in DES-DES and 7 proteins in DES-Tx OVX mice were different from those in Oil-DES and Oil-Tx OVX mice, respectively. These findings indicate that neonatal DES treatment alters the behavior of the vagina and uterus in response to postpubertally administered DES and Tx.

The distribution of the estrogen receptor (ER) was investigated immunohistochemically in the genital tract of 0 (day of birth)--to 50-day-old C57BL/Tw female mice given 5 daily injections of 2 micrograms diethylstilbestrol (DES) from the day of birth using an anti-ER monoclonal antibody. In the oviduct, cervix and vagina of control mice, both epithelial and stromal cell nuclei showed positive ER immunoreaction at 0 day. Nuclei of uterine stromal cells also showed strong ER immunostaining even at 0 day. In contrast, the ER in uterine epithelial cells began to appear by day 5. The number of positive epithelial cells and staining intensity gradually increased until 10 days. In neonatally DES-exposed mice, ER appeared in uterine epithelial cells 24 h after the injection. Polygonal cells appearing in the vaginal epithelium of DES-exposed 3-day-old mice showed weak ER staining. Neonatal DES exposure reduced staining intensity of columnar vaginal epithelial cells and uterine stromal cells at 3 days. Vaginal epithelial cells undergoing ovary-independent stratification and cornification in neonatally DES-exposed mice and normal estrous mice showed ER immunoreaction only in the basal cells.

Neonatal treatment of female mice with diethylstilbestrol (DES) results in genital tract abnormalities including ovary-independent vaginal proliferation and cornification. Protein profiles were examined in vagina and uterus from 45-day-old, ovariectomized C57BL/Tw mice which had been given 5 daily injections of 2 micrograms DES or oil vehicle alone from the day of birth, and in those from 45-day-old, ovariectomized mice given 3 daily injections of 0.1 microgram DES from 42 days of age. Proteins extracted were analyzed by two-dimensional polyacrylamide gel electrophoresis. In the vagina, expression of 37 non-keratin proteins was altered by postpubertal injections of DES as compared with the controls. In the neonatally DES-exposed vagina, expression of 26 of 37 proteins was altered as compared with controls. In the uterus, expression of 22 proteins was altered in the postpubertally DES-exposed group as compared with that in the control; however, the protein expression pattern of the neonatally DES-exposed group closely resembled that of the control except for one protein (no. 23, pI = 6.1, MW = 39.5 kDa) which was specifically increased in the neonatally DES-exposed group. By immunoblot analysis, 6 keratin polypeptides (49.5, 50, 52, 53, 57 and 58 kDa) were identified in vaginae of ovariectomized mice exposed neonatally and postpubertally to DES and of the controls. These results indicate that neonatal DES exposure induces organ specific alterations in the synthesis of proteins in mouse vagina and uterus.

In 35-day-old female ICR/JCL mice given 5 daily injections of 1 microgram diethylstilbestrol (DES) from the day of birth, a significantly higher incidence of polyovular follicles was found in the ovaries than in those of age-matched control mice. Gap junctions of granulosa cells of mature follicles in neonatally DES-exposed mice were larger than those of the controls. When stimulated by gonadotropins (PMSG and hCG) and caged with males, the number of tubal embryos in DES-exposed mice was less, but the rate of fertilization and development was not different compared to the controls. Division of oocytes collected from the ovaries of 40-day-old DES-exposed and control mice after stimulation of gonadotropins was examined 24 to 72 h after in vitro insemination to ascertain whether fertilization had occurred in oocytes from polyovular follicles. Seventy-seven % of oocytes from uniovular follicles of control mice developed up to 8-cell stage embryos following in vitro insemination; 66% of those from similar follicles of DES-exposed mice developed into the same stage. By contrast, only 47% of oocytes from polyovular follicles of DES-exposed mice showed the division up to 8-cell stage 72 h after insemination, indicating a significantly lower fertilization rate compared to the oocytes from uniovular follicles of control and DES-exposed mice. Without insemination, oocytes taken from the same pools in these experiments never divided during the period of manipulation and incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Genital organs of C57BL/Tw female mice given 5 daily injections of 100 micrograms tamoxifen (Tx) from the day of birth (day 0) were examined at 5, 10, 15, 20, 30 and 60 days of age. The incidence of polyovular follicles (PF) in Tx mice was higher, but the development of uterine glands and tunica muscularis uteri in Tx mice was lower than in the age-matched controls. Uteri of 15-day-old Tx mice underwent a weight increase resulting from edematous change in the stromal tissue lacking type I collagen and fibronectin. Adenosis-like lesions were found in the vaginae of 5- to 30-day-old Tx mice. Mice given neonatal injections of 100 micrograms clomiphene (Clm) and nafoxidine (Naf) were also examined at 60 days. Tx caused much greater damage to the ovary and uterus than did Clm and Naf. In order to examine the critical period of induction by Tx of female genital organ abnormalities, mice were given 5 daily injections of Tx starting at different early postnatal ages. Tx injections starting within 5 days caused a high incidence of PF in the ovary and aplasia of tunica muscularis in the uterus. The Tx treatment also induced atrophy of the uterine luminal epithelium when started within 7 days. The present study suggests, therefore, that the postnatal limit of the critical period for the female genital organs lies within 7 days after birth.